The objective of this research is to understand how gene expression is regulated temporally and spatially in bacteria. Sporulation of Bacillus subtilis provides an attractive experimental system to elucidate novel mechanisms of signaling and gene regulation likely to be used by other bacteria, including pathogens. During sporulation, the cell is divided into mother cell and forespore compartments, each with a copy of the genome. Different sigma subunits of RNA polymerase (RNAP) become active in each compartment at different times, accounting for much of the temporal and spatial gene regulation. In addition, transcription factors activate or repress expression of many genes. This proposal focuses on mechanisms of gene regulation in the mother cell compartment, where two sigma factors and two transcription factors are organized in a hierarchical cascade with the order sigmaE, SpoIIID, sigmaK, then GerE. Activity of each sigma is governed by a unique signaling pathway that begins in the forespore and ends with proteolytic removal of an inhibitory pro-sequence in the mother cell. Components of these signaling pathways are known, but the molecular mechanisms of signaling and proteolytic processing remain to be elucidated. One aim is to understand the mechanism of pro-sigmaK processing and its regulation by BofA. This pathway involves regulated intramembrane proteolysis (RIP) of pro-sigmaK by SpoIVFB metalloprotease. RIP is involved in crucial biological processes, but is poorly understood. RIP of pro-sigmaK by SpoIVFB provides a fantastic opportunity to break new ground. Processing of pro-sigmaE does not appear to involve RIP, but its mechanism and its regulation by a signal from the forespore are unclear. To clarify this pathway, approaches used successfully to study pro-sigmaK processing will be applied. Another aim is to investigate the importance of a feedback loop by which sigmaK RNAP negatively regulates early gene expression. This will give insight into the switch from one regulon to another, a common feature in bacterial and phage gene regulation. Finally, SpoIIID's small size and essential role in sporulation make it attractive for structure/function and genetic analyses that will add knowledge about mechanisms of transcriptional activation in bacteria.